The SNARE protein syntaxin 5 exists as long (42 kDa) and short (35 kDa) isoforms. The short form is principally localized in the Golgi complex, whereas the long form resides not only in the Golgi but also in the endoplasmic reticulum (ER). Although the Golgi-localized short form has been extensively investigated, little is known about the long form. In the present study, we demonstrate that the long form of syntaxin 5 functions to shape the ER. We found that overexpression of the long form of syntaxin 5 induces rearrangement and co-alignment of the ER membrane with microtubules, the pattern of which is quite similar to that observed in cells overexpressing CLIMP-63, a linker between the ER membrane and microtubules. The ability of syntaxin 5 to induce ER-microtubule rearrangement is not related to its SNARE function, but correlates with its binding affinities for CLIMP-63, and CLIMP-63 is essential for the induction of this rearrangement. Microtubule co-sedimentation assays demonstrated that the long form of syntaxin 5 has a substantial microtubule-binding activity. These results suggest that the long form of syntaxin 5 contributes to the regulation of ER structure by interacting with both CLIMP-63 and microtubules. Indeed, depletion of syntaxin 5 caused the spreading of the ER to the cell periphery, similar to the phenotype observed in cells treated with the microtubule-depolymerizing reagent nocodazole. Our results disclose a previously undescribed function of the long form of syntaxin 5 that is not related to its function as a SNARE.